[unreadable] The overall aim of this proposal is to develop approaches for repetitive non-invasive in vivo imaging of the fate of T lymphocytes after their adoptive transfer for anti-tumor vaccination. The proposed imaging approaches will allow to answer several questions related to T-cell migration and homing to the tumor target, their subsequent activation and cytolytic activity, and the duration of T-cell viability. The ultimate aim of the proposed imaging approaches is to assess the degree of T cell activation at tumor target, to assess the cytolitic potential of tumor infiltrating T cells, and to predict their tumorolytic efficacy early on during therapy. We propose to combine the genetic labeling of T-cells with multiple reporter genes followed by the repetitive administration of short-lived radioisotope reporter probes to sequentially image the reporter gene expressing cells with PET or other imaging technologies (i.e., fluorescence, bioluminescence, MRI). Specifically, we will explore the feasibility of genetic labeling of T-cells with novel reporter gene - reporter probe combinations and novel multi-reporter gene constructs. We will test the feasibity of a novel reporter gene, the truncated human mitochondrial thymidine kinase type two (hTK2) using [124I]FIAU and newly developed short lived radiolabeled probes for imaging of hTK2 [2'-18F]FEAU and [11C]FEAU. We plan to develop several dual-reporter systems in which the hTK2 will be expressed constitutively as a "beacon" reporter gene and the mutant HSV1-tk (HSV1-sr39tk) gene (imaged by [18F]FHBG) will be expressed as "sensor" reporter gene in a T cell- and activation-specific manner. The proposed multi-reporter gene imaging paradigm will be implemented in the assessment and optimization of new anti-cancer T-cell vaccines that are based on the artificial anti-CEA or anti-PSMA T-cell receptors and used for treatment of colorectal and prostate carcinomas, respectively. Imaging will be used for monitoring the trafficking, localization and activation of adoptively transferred T cells before and during co-stimulatory therapy with various cytokines. These studies will assess the feasibility and applicability of the proposed therapeutic and imaging approaches for implementation in clinical setting. [unreadable] [unreadable]